Engineered Cyclic Peptides

ABSTRACT

An engineered cyclic peptide provides structural constraints to resist non-specific degradation in the human body and includes environment-specific cleavage sites to allow release of a linearized peptide upon reaching a target environment. The linearized peptide can include a reporter molecule or a bioactive therapeutic such that the cyclic peptide is essentially inactive at administration and in circulation but becomes reactive only upon exposure to target-specific environmental factors such as a specific combination of differentially-expressed proteases associated with a target tissue or disease state. The peptides can include tuning that modulate distribution by targeting the particle to specific tissue, bodily fluids, or cell types.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalApplication No. 62/682,492, filed Jun. 8, 2018, the content of which isincorporated by reference.

TECHNICAL FIELD

The invention relates to engineered cyclic peptides with target orprotease-specific cleavage sites.

BACKGROUND

New mechanisms for diagnosing, monitoring, and treating seriousdiseases, such as cancer, continue to evolve and offer new hope forpatients. Some promising methods rely on uptake and residence in targetareas (e.g., specific tissues or a tumor microenvironment) in order toachieve a therapeutic or diagnostic result. However, a major problemwith those types of approaches is that the diagnostic or therapeuticentities are typically degraded, at least in part, before they arrive atthe tissue environment in which they are intended to act. Peptides areparticularly susceptible to degradation.

Additionally, some diagnostic and therapeutic molecules have thepotential for undesirable reactions with off-target tissues orenvironments or have functionalities that depend on reacting only with aspecific target tissue or environment.

Accordingly, many promising diagnostic and treatment methods remainimpractical to apply due to the inability to reach a target environmentin the body without degrading or otherwise having off-target reactions.

SUMMARY

The invention provides cyclic peptides that are resistant to degradationin the body while in transit to a target environment. Cyclic peptidesprovide structural constraints that resist degradation by, for example,proteases in the blood. The cyclic peptides are engineered with one ormore target-specific cleavage sites such that, upon arriving in thetarget environment, they are cleaved to release a molecule that isreactive in the target environment.

In one example, the target environment may be a tumor microenvironmentin which a specific enzyme or set of enzymes aredifferentially-expressed. According to the invention, a cyclic peptideis engineered with cleavage sites specific to enzymes in the tumor(e.g., unique enzymes expressed preferentially in the tumor). Theengineered peptide, in its cyclic form, can travel through the blood andother potentially harsh environments protected against degradation bycommon non-specific proteases and without interacting in a meaningfulway with off-target tissues. Only upon arrival within the specifictarget tissue and exposure to the required enzyme or combination ofenzymes, the cyclic peptide is cleaved to produce a linear molecule thatis capable of carrying out a diagnostic or therapeutic function. Forpurposes of the application and as will be apparent upon considerationof the detailed description thereof, a linear peptide is any peptidethat is not cyclic. Thus, for example, a linearized peptide may havevarious branch chains.

In various embodiments, a linearized peptide of the invention comprisesa reporter molecule or a therapeutic molecule such that reaction withthe environment includes releasing a detectable reporter to diagnose ormonitor a disease state or releasing a bioactive compound such as atherapeutic peptide operable to treat the target tissue.

Cyclic peptides can be engineered with other cleavable linkages, such asester bonds in the form of cyclic depsipeptides in which the degradationof the ester bond releases a linearized peptide ready to react with itstarget environment. Thioesters and other tunable bonds can be includedin the cyclic peptide to create a timed-release in plasma or otherenvironments. See Lin and Anseth, 2013 Biomaterials Science (ThirdEdition), pages 716-728, incorporated herein by reference.

According to the invention, cyclic peptides may release a reportermolecule upon linearization and cleavage of a specific cleavage sitethat is the target of a unique enzyme in the disease microenvironment.For example, a cyclic peptide may include a polyethylene glycol (PEG)scaffold and one or more polypeptide reporters. The cleavable links inthe cyclic peptide are specific for different enzymes whose activity ischaracteristic of a condition of tissue. When administered to a patient,the cyclic peptide can locate to a target tissue, where it is linearizedby the enzymes to release the detectable analytes. The analytes are thendetected in a patient sample, such as urine. The detected analytes serveas a report of which enzymes are active in the tissue.

Because enzymes are differentially expressed under the physiologicalstate of interest, such as a disease stage or degree of diseaseprogression, analysis of the sample provides a non-invasive test for thephysiological state (e.g., disease stage or condition) of the organ,bodily compartment, bodily fluid, or tissue. Due to the protectionagainst off-target degradation afforded by cyclic peptides of theinvention, false-positives are reduced as reporter release and detectionis more likely to have occurred due to differential-expression ofenzymes specific to a certain disease or physiologic state.False-negatives are also reduced as the cyclic peptide is more likely toremain intact until arriving at the target tissue in order to performits diagnostic role in the presence of the target-specific enzymes.

Macrocyclic peptides may contain two or more protease-specific cleavagesequences and can require two or more protease-dependent hydrolyticevents to release a reporter peptide or a bioactive compound. Theprotease-specific sequences can be different in various embodiments. Incases where cleavage of multiple sites is required to release thelinearized peptide, different protease-specific sequences can increasespecificity for the release as the presence of at least two differenttarget-specific enzymes will be required. In other embodiments, multipledifferent cleavage sites may be provided where cleavage of any singlesite will release the linearized peptide. In such instances, a singlepeptide can be tuned to linearize and interact with a variety ofenvironments for two or more different targets. The specific andnon-specific proteolysis susceptibility and rate can be tuned throughmanipulation of peptide sequence content, length, and cyclizationchemistry.

Cyclic peptides of the invention may include a carrier molecule. Carrierstructure can include multiple molecular subunits and may be, forexample, a multi-arm polyethylene glycol (PEG) polymer, a lipidnanoparticle, or a dendrimer or a peptide sequence. The detectableanalytes or reporters in diagnostic embodiments may be, for example,polypeptides that are cleaved by proteases that are differentiallyexpressed in tissue or organs under a specified physiological state,e.g., affected by disease. Because the carrier structure and thedetectable analytes are biocompatible molecular structures that locateto a target tissue and are cleaved by disease-associated enzymes torelease analytes detectable in a sample, compositions of the disclosureprovide non-invasive methods for detecting and characterizing a diseasestate or stage of an organ or tissue. Because the compositions providesubstrates that are released as detectable analytes by enzymaticactivity, quantitative detection of the analytes in the sample provide ameasure of rate of activity of the enzymes in the organ or tissue. Thusmethods and compositions of the disclosure provide non-invasivetechniques for measuring both stage and rate of progression of a diseaseor condition in a target organ or tissue.

Additionally, the cyclic peptides may include additional molecularstructures to influence trafficking of the peptides within the body, ortiming of the enzymatic cleavage or other metabolic degradation of theparticles. The molecular structures may function as tuning domains,additional molecular subunits or linkers that are acted upon by the bodyto locate the cyclic peptide to the target tissue under controlledtiming. For example, the tuning domain may target the particle tospecific tissue or cell types. Trafficking may be influenced by theaddition of molecular structures in the carrier polymer by, for example,increasing the size of a PEG scaffold to slow degradation in the body.

In certain embodiments, the invention provides a tunable cyclic peptidethat reveals enzymatic activity associated with a physiological state,such as disease. When the activity reporter is administered to apatient, it is trafficked through the body to specific cells or specifictissues. Alternatively, the sensor may be designed or tuned so that itremains in circulation, e.g., in blood, or lymph, or both. If enzymesthat are differentially expressed under conditions of a particulardisease are present, those enzymes cleave the reporter and release adetectable analyte. The cyclic nature of the peptides may be used toresist non-specific degradation of the peptide in circulation whilestill providing an accessible substrate for cleavage by the targetproteases.

Where the cleavage sites are specific to enzymes known to be active intissue affected by a disease, detection of the analyte is indicative ofthe disease condition. For example, when the peptide includes cleavagetargets of proteases expressed in liver fibrosis, the cyclic peptide iscleaved in the liver to release the detectable analyte into circulationafter which renal filtration excretes the detectable analyte in urine.Presence of the analyte in a urine sample from the patient is asignature of liver fibrosis in the patient.

Molecular structures can be included in the cyclic peptide as tuningdomains, to tune or modify a distribution or residence time of thecyclic peptide within the subject. The tuning domains may be linked anyportion of the cyclic peptide and may be modified in numerous ways.Through the use of tuning domains, one may modify the cyclic peptide'sdistribution within the body depending on in vivo trafficking pathwaysto a specific tissue, or its residence time within systemic circulationor within a specific tissue. Additionally, the tuning domains maypromote effective cleavage of the reporter by tissue-specific enzymes orprevent premature cleavage or hydrolysis.

Cyclic peptides according to the invention provide sensitive, specific,and non-invasive method for detecting disease-associated activity thatare able to persist until reaching a target environment and minimizeoff-target cleavage and associated false positive indications. Thecyclic peptides are acted upon in the body of the patient so that thedetectable analyte is released in such a manner as to indicate criticaldisease states at a very early stage. The cyclic peptides may includeadditional molecular structures as tuning domains that employ the bodyfor sample preparation by presenting a molecular complex that onlyreleases the detectable analyte into a collectable sample when the bodyprocesses the cyclic peptide in a detectable manner. The tuning domains,which may be included within the peptide structure to modulate proteasecleavage, allow for precise tuning of the biological fate of the cyclicpeptide. Additionally, because the detectable analytes are the productof enzymatic activity and the cyclic peptides can be provided in excess,the signal given by the analyte is effectively amplified, and thepresence of even very small quantity of active enzyme may be detected.Because the tuning domains can target the cyclic peptide to specifictissue of the body and because the reporter is known to be cleaved byenzymes associated with a disease, the cyclic peptides can provide forvery rapid and sensitive disease detection.

Aspects of the invention include an engineered cyclic peptide with oneor more cleavage sites cleavable within a target environment, whereincleavage of the one or more cleavage sites releases a linearized peptidereactive with the target environment. In various embodiments, the targetenvironment may be a tumor or a biological fluid such as blood. Anyenvironment with a distinguishable characteristic (e.g. a specific pH orcombination of proteases) that a cleavage site can be tuned to can betargeted using cyclic peptides of the invention.

The cleavage site may be cleaved by an enzyme present in the targetenvironment. The enzyme may be known to be expressed with a certaindisease or medical condition and the linearized peptide can be atherapeutic peptide operable to treat the disease or medical condition.The linearized peptide may be bioactive within the target environment.In various embodiments, the cyclic peptide may be a cyclic depsipeptideand the one or more cleavage sites can comprise an ester bond. In someembodiments, the cyclic peptide can be a macrocyclic peptide with acyclization chemistry other than ester bonds.

Cyclic peptides of the invention can include a carrier. The carrier caninclude a poly ethylene glycol (PEG) scaffold of covalently linked PEGsubunits. As noted above, the linearized peptide can include adetectable reporter. The detectable reporter may include a volatileorganic compound, an elemental mass tag, a peptide comprising one ormore D-amino acids, a nucleic acid, or a neoantigen. The detectablereporter may include an elemental mass tag comprising an element ofatomic number greater than 20.

In certain embodiments, the reporter can include an antigen detectableby a hybridization assay. The reporter can include a fluorescent donorsuch as a carboxyfluorescein (FAM) and the cyclic peptide may include aquencher such as CPQ₂ (available from CPC Scientific, Sunnyvale, Calif.)such that target-specific cleavage of the cyclic peptide results indequenching of the fluorescent donor for subsequent detection. The oneor more cleavage sites may comprise a plurality of different cleavagesites which may be cleaved by different enzymes. Cleavage of two or moreof the plurality of different cleavage sites may be required to releasethe linearized peptide. The two or more of the plurality of differentcleavage sites may be required to be cleaved in a specific order torelease the linearized peptide.

In various embodiments, cyclic peptides may include a tuning domain thatmodifies a distribution or residence time of the engineered cyclicpeptide within a subject when administered to the subject. Tuningdomains can include ligands for receptors of a specific cell or aspecific tissue type to target the cyclic peptide to the target cell ortissue type. The ligands may promote accumulation of the engineeredcyclic peptide in the specific tissue type or body compartment and caninclude a small molecule, a peptide, an antibody, a fragment of anantibody, a nucleic acid, or an aptamer. The tuning domains can includehydrophobic chains that facilitate diffusion of the engineered cyclicpeptide across a cell membrane. Aspects of the invention include methodsof preparing or administering cyclic peptides as described herein to asubject for the diagnosis, monitoring, or treatment of a disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 diagrams steps of a method for designing a cyclic peptide.

FIG. 2 shows an engineered macrocyclic peptide.

FIG. 3 shows protease-specific of an engineered macrocyclic peptide

FIG. 4 shows protease-specific cleavage of a double gated engineeredmacrocyclic peptide.

FIG. 5 shows a controlled-release cyclic peptide.

FIG. 6 shows of linearization of a cyclic depsipeptide throughhydrolysis of the ester bond followed by protease-specific cleavage.

FIG. 7 shows activity detection according to certain embodiments.

FIG. 8 shows a bicyclic peptide with 3 cleavage substrates.

FIG. 9 shows an exemplary cleavage of a bicyclic peptide.

FIG. 10 shows a tricyclic peptide with 4 cleavage substrates.

FIG. 11 shows a polycyclic peptide with 4 cyclic structures and 5cleavage substrates.

FIG. 12 shows a polycyclic peptide with an even number (n) of cyclicstructures and n+1 cleavage substrates.

FIG. 13 shows a polycyclic peptide with an odd number (n) of cyclicstructures and n+1 cleavage substrates.

DETAILED DESCRIPTION

The invention provides cyclic peptides that are structurally resistantto non-specific proteolysis and degradation in the body. Cyclic peptidesof the invention include environment-specific cleavage sites such asprotease-specific substrates or pH-sensitive bonds that allow theotherwise non-reactive cyclic peptide to release a reactive linearizedpeptide. Those elements allow the peptides, carrying diagnostic reporteror therapeutic molecules to reach their intended target tissue orenvironment intact to then carry out their intended purpose at thetarget. The inclusion of target-environment-specific cleavage sitesallows for highly selective targeting for diagnostic reporting andtherapeutic delivery. Cyclic peptides can include tuning domains tomodify distribution and residence times in various tissues orenvironments within the body.

Cyclic peptides can require cleavage at a plurality of cleavage sites toincrease specificity. The plurality of sites can be specific for thesame or different proteases. Polycyclic peptides can be used comprising2, 3, 4, or more cyclic peptide structures with various combinations ofenzymes or environmental conditions required to linearize or release thefunctional peptide or other molecule. Cyclic peptides can includedepsipeptides wherein hydrolysis of one or more ester bonds release thelinearized peptide. Such embodiments can be used to tune the timing ofpeptide release in environments such as plasma.

Macrocyclic peptides occur naturally and have been studied and prizedfor their resistance to degradation by proteases generally present inblood. See Gang, et al., 2018, Cyclic Peptides: Promising Scaffolds forBiopharmaceuticals, Genes, 9:557, incorporated herein by reference.Cyclization of peptides has also been shown to facilitate passagethrough cell membrane allowing access to both extra and intracellulartargets and, due to their promising attributes, several approaches fordesigning and producing synthetic cyclic peptides are known. Id.

Cyclic peptides of the invention may include a carrier, a therapeuticpeptide, a reporter and one or more tuning domains that modifies adistribution or residence time of the cyclic peptide (or a releasedlinearized peptide reporter or therapeutic) within a subject whenadministered to the subject. The cyclic peptide may be designed todetect and linearize in response to any enzymatic activity in the body,for example, enzymes that are differentially expressed under aphysiological state of interest such as dysregulated protease activityindicative of a disease state. Dysregulated proteases have importantconsequences in the progression of diseases such as cancer in that theymay alter cell signaling, help drive cancer cell proliferation,invasion, angiogenesis, avoidance of apoptosis, and metastasis.

The cyclic peptide may be tuned via the tuning domains in numerous waysto facilitate responsiveness to enzymatic activity within the body inspecific cells or in a specific tissue. For example, the cyclic peptidemay be tuned to promote distribution of the cyclic peptide to thespecific tissue or to improve a residence time of the cyclic peptide inthe subject or in the specific tissue.

When administered to a subject, the cyclic peptide is trafficked throughthe body and may diffuse from the systemic circulation to a specifictissue, where the peptide may be cleaved via enzymes indicative of thedisease to release a detectable analyte or a therapeutic compound. Inthe case of a reporter molecule, the detectable analyte may then diffuseback into circulation where it may pass renal filtration and be excretedinto urine, whereby detection of the detectable analyte in the urinesample indicates enzymatic activity upon the reporter.

The carrier may be any suitable platform for trafficking the moleculesthrough the body of a subject, when administered to the subject. Thecarrier may be any material or size suitable to serve as a carrier orplatform. Preferably the carrier is biocompatible, non-toxic, andnon-immunogenic and does not provoke an immune response in the body ofthe subject to which it is administered. The carrier may also functionas a targeting means to target the cyclic peptide to a tissue, cell ormolecule. In some embodiments the carrier domain is a particle such as apolymer scaffold. The carrier may, for example, result in passivetargeting to tumors or other specific tissues by circulation. Othertypes of carriers include, for example, compounds that facilitate activetargeting to tissue, cells or molecules. Examples of carriers include,but are not limited to, nanoparticles such as iron oxide or goldnanoparticles, aptamers, peptides, proteins, nucleic acids,polysaccharides, polymers, antibodies or antibody fragments and smallmolecules.

The carrier may include a variety of materials such as iron, ceramic,metallic, natural polymer materials such as hyaluronic acid, syntheticpolymer materials such as poly-glycerol sebacate, and non-polymermaterials, or combinations thereof. The carrier may be composed in wholeor in part of polymers or non-polymer materials, such as alumina,calcium carbonate, calcium sulfate, calcium phosphosilicate, sodiumphosphate, calcium aluminate, and silicates. Polymers include, but arenot limited to: polyamides, polycarbonates, polyalkylenes, polyalkyleneglycols, polyalkylene oxides, cellulose ethers, cellulose esters, nitrocelluloses, polymers of acrylic and methacrylic esters, methylcellulose, ethyl cellulose, and hydroxypropyl cellulose. Examples ofnon-biodegradable polymers include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

Examples of biodegradable polymers include synthetic polymers such aspolymers of lactic acid and glycolic acid, poly-anhydrides,polyurethanes, and natural polymers such as alginate and otherpolysaccharides including dextran and cellulose, collagen, albumin andother proteins, copolymers and mixtures thereof. In general, thesebiodegradable polymers degrade either by enzymatic hydrolysis orexposure to water in vivo, by surface or bulk erosion. Thesebiodegradable polymers may be used alone, as physical mixtures (blends),or as co-polymers.

In preferred embodiments, the carrier includes biodegradable polymers sothat whether the reporter is cleaved from the carrier, the carrier willbe degraded in the body. By providing a biodegradable carrier,accumulation and any associated immune response or unintended effects ofintact cyclic peptides remaining in the body may be minimized.

Other biocompatible polymers include PEG, PVA and PVP, which are allcommercially available. PVP is a non ionogenic, hydrophilic polymerhaving a mean molecular weight ranging from approximately 10,000 to700,000 and has the chemical formula (C6H9NO)[n]. PVP is also known aspoly[1(2 oxo 1 pyrrolidinyl)ethylene]. PVP is nontoxic, highlyhygroscopic and readily dissolves in water or organic solvents.

Polyvinyl alcohol (PVA) is a polymer prepared from polyvinyl acetates byreplacement of the acetate groups with hydroxyl groups and has thechemical formula (CH2CHOH)[n]. Most polyvinyl alcohols are soluble inwater.

Polyethylene glycol (PEG), also known as poly(oxyethylene) glycol, is acondensation polymer of ethylene oxide and water. PEG refers to acompound that includes repeating ethylene glycol units. The structure ofPEG may be expressed as H—(O—CH2-CH2)n-OH. PEG is a hydrophilic compoundthat is biologically inert (i.e., non-immunogenic) and generallyconsidered safe for administration to humans.

When PEG is linked to a particle, it provides advantageous properties,such as improved solubility, increased circulating life, stability,protection from proteolytic degradation, reduced cellular uptake bymacrophages, and a lack of immunogenicity and antigenicity. PEG is alsohighly flexible and provides bio-conjugation and surface treatment of aparticle without steric hindrance. PEG may be used for chemicalmodification of biologically active compounds, such as peptides,proteins, antibody fragments, aptamers, enzymes, and small molecules totailor molecular properties of the compounds to particular applications.Moreover, PEG molecules may be functionalized by the chemical additionof various functional groups to the ends of the PEG molecule, forexample, amine-reactive PEG (BS (PEG)n) or sulfhydryl-reactive PEG (BM(PEG)n).

In certain embodiments, the carrier is a biocompatible scaffold, such asa scaffold including polyethylene glycol (PEG). In a preferredembodiment, the carrier is a biocompatible scaffold that includesmultiple subunits of covalently linked poly(ethylene glycol) maleimide(PEG-MAL), for example, an 8-arm PEG-MAL scaffold. A PEG-containingscaffold may be selected because it is biocompatible, inexpensive,easily obtained commercially, has minimal uptake by thereticuloendothelial system (RES), and exhibits many advantageousbehaviors. For example, PEG scaffolds inhibit cellular uptake ofparticles by numerous cell types, such as macrophages, which facilitatesproper distribution to a specific tissues and increases residence timein the tissue.

Cleavage of the cyclic peptide is preferably dependent on enzymes thatare active in a specific disease state. For example, tumors areassociated with a specific set of enzymes. For a tumor, the cyclicpeptide may be designed with one or more enzyme susceptible sites thatmatch the enzymes expressed by the tumor or other diseased tissue.

In various embodiments, the cyclic peptide may include a reportercomprising a naturally occurring molecule such as a peptide, nucleicacid, a small molecule, a volatile organic compound, an elemental masstag, or a neoantigen. In other embodiments, the reporter includes anon-naturally occurring molecule such as D-amino acids, syntheticelements, or synthetic compounds. The reporter may be a mass-encodedreporter, for example, a reporter with a known andindividually-identifiable mass, such as a polypeptide with a known massor an isotope.

An enzyme may be any of the various proteins produced in living cellsthat accelerate or catalyze the metabolic processes of an organism.Enzymes act on substrates. The substrate binds to the enzyme at alocation called the active site before the reaction catalyzed by theenzyme takes place. Generally, enzymes include but are not limited toproteases, glycosidases, lipases, heparinases, phosphatases. Examples ofenzymes that are associated with disease in a subject include but arenot limited to MMP, MMP-2, MMP-7, MMP-9, kallikreins, cathepsins,seprase, glucose-6-phosphate dehydrogenase (G6PD), glucocerebrosidase,pyruvate kinase, tissue plasminogen activator (tPA), a disintegrin andmetalloproteinase (ADAM), ADAM9, ADAM15, and matriptase.

Examples of substrates for disease-associated enzymes include but arenot limited to Interleukin 1 beta, IGFBP-3, TGF-beta, TNF, FASL, HB-EGF,FGFR1, Decorin, VEGF, EGF, IL2, IL6, PDGF, fibroblast growth factor(FGF), and tissue inhibitors of MMPs (TIMPs).

The disease or condition targeted by the cyclic peptide may be anydisease or condition that is associated with an enzymatic activity. Forexample, cancer progression and metastasis, cardiovascular disease,liver fibrosis, nonalcoholic fatty liver disease (NAFLD), arthritis,viral, bacterial, parasitic or fungal infection, Alzheimer's diseaseemphysema, thrombosis, hemophilia, stroke, organ dysfunction, anyinflammatory condition, vascular disease, parenchymal disease, or apharmacologically-induced state are all known to be associated withenzymatic activity.

The tuning domains may include any suitable material that modifies adistribution or residence time of the cyclic peptide within a subjectwhen the cyclic peptide is administered to the subject. For example, thetuning domains may include PEG, PVA, or PVP. In another example, thetuning domains may include a polypeptide, a peptide, a nucleic acid, apolysaccharide, volatile organic compound, hydrophobic chains, or asmall molecule.

FIG. 1 diagrams steps of a method 100 for designing a target-specificcyclic peptide. At step 105, gene expression in subjects with a knowndisease may be determined, for example, by performing RNA sequencing(RNA-Seq) on gene transcripts using a next-generation sequencingplatform, and determining fold-change in expression level of atranscript associated with the disease by normalizing read counts fromthe measured transcripts against healthy control read counts.

At step 110, for example, gene expression that is upregulated inrelation to a non-diseased state may be determined, for example, toidentify candidate proteases indicative of a disease. By identifyingcandidate proteases indicative of the disease, associated proteasesubstrates may also be identified and incorporated into the reporter ofthe cyclic peptide.

At step 115, a cyclic peptide is assembled which may include a carrier,one or more tuning domains and a reporter and/or a therapeutic peptide.

The cyclic peptide can include one or more cleavage sites susceptible tothe protease activity as identified in step 110. Tuning domains may bepresent in or linked to the cyclic peptide and may be based on the invivo pathway through which the cyclic peptide is to be trafficked orbased on the intended method of detection in the case of reporter-linkedtuning domains. For example, the tuning domains may include PEG and canbe linked to the peptide to facilitate distribution to the liver todetect protease activity in the liver, and the reporter may be detectedvia a ligand binding assay, such as an ELISA assay. The cyclic peptidemay include any reporter or therapeutic peptide. The therapeutic peptidemay be one operable to treat the disease associated with the upregulatedgenes identified in step 110.

At step 120, the cyclic peptide may be administered to a subject havingthe disease for targeted delivery of a therapeutic to diseased tissue orto detect enzymatic activity indicative of the disease, such asdysregulated protease activity.

The cyclic peptides may be administered by any suitable method ofdelivery. In preferred embodiments, the cyclic peptide is deliveredintravenously or aerosolized and delivered to the lungs, for example,via a nebulizer. In other examples, the cyclic peptide may beadministered to a subject transdermally, intradermally, intraarterially,intralesionally, intratumorally, intracranially, intraarticularly,intratumorally, intramuscularly, subcutaneously, orally, topically,locally, inhalation, injection, infusion, or by other method or anycombination known in the art (see, for example, Remington'sPharmaceutical Sciences (1990), incorporated by reference).

At step 125, in the case of the cyclic peptide including a reportermolecule released by its cleavage, the target enzymatic activity may bedetected in any biological sample. In preferred embodiments, thebiological sample is non-invasively obtained and is preferably a bodilyfluid or other substance that is naturally excreted from the body.

FIG. 2 shows an exemplary cyclic peptide 201 having a protease-specificsubstrate 209 and a stable cyclization linker 203. The N-terminus andC-terminus, coupled to the cyclization linker 203 comprise cyclizationresidues 205. The peptide may be engineered to address considerationssuch as protease stability, steric hindrance around cleavage site,macrocycle structure, and rigidity/flexibility of peptide chain. Thetype and number of spacer residues 207 can be chosen to address andalter many of those properties by changing the spacing between thevarious functional sites of the cyclic peptide. The cyclization linkerand the positioning and choice of cyclization residues can also impactthe considerations discussed above. Tuning domains such as PEG and/orreporters such as FAM can be included in the cyclic peptide.

FIG. 3 shows protease-specific cleavage of a macrocyclic peptide of theinvention. The macrocyclic peptide is resistant to degradation duringgeneral circulation and in the presence of non-specific proteases it mayencounter in off-target tissue. The macrocyclic peptide shown includestwo protease-specific substrates and cleavage of both is required torelease the linearized reporter molecule. In FIG. 3, both cleavage sitesare the same and, accordingly, exposure to the protease in the targetenvironment results in cleavage of both substrates which fully separatesthe quencher (CPQ₂) from a fluorescent reporter (FAM) producing adetectable signal indicative of the presence of the specific proteasewhich, in turn, can be indicative of the presence of a disease. Theunquenched fluorescent reporter or other reporter molecule may be linkedto a tuning domain operable to promote concentration of the linkedreporter molecule in the biological sample (e.g., urine) for detection.

In other embodiments, as depicted in FIG. 4, the substrates may bespecific for different proteases creating a double-gated substratewherein exposure to both proteases is required for the release of thelinearized peptide (in the case of FIG. 4 a dequenched carboxyfluorescein reporter). The requirement of two or more differentproteases can help further tune the cyclic peptide specificity which canbe especially useful in instances where the upregulated proteasesindicative of disease are individually common but present a more uniquecombination.

FIG. 5 shows a cyclic peptide 501 according to certain embodiments. TheC-terminus and N-terminus of the peptide include cyclization residues505 that are linked together by a cyclization linker 503. The peptide501 is made as stable as possible to proteolysis but the cyclizationlinker 503 may be unstable overtime or otherwise responsive to themicroenvironment of a targeted tissue (e.g., via redox state, pH, orpresence of other enzymes). For example, as shown in FIG. 6, thecyclization linker 503 may comprise an ester bond having a knownhydrolysis rate in plasma for a controlled degradation and release ofthe linearized, reactive peptide. The cyclic peptide 501 includes aprotease-specific substrate 509 such that both a time-specific cleavageevent (e.g., hydrolysis of the ester bond) as well as anprotease-specific cleavage event (e.g., by a disease orenvironment-specific upregulated enzyme) are required to release thefunctional linearized peptide. Various cross linking within the peptidesequence to modify performance and response in the subject is possiblein certain embodiments.

In certain embodiments, the substrate may be conformationally blockedfrom proteolytic interactions while in a cyclic form such thatprotease-specific cleavage of the substrate is impossible until theunstable cyclization linker has degraded. In such a manner, reporter ortherapeutic release reactions can be delayed until the cyclic peptidehas had time to localize in the target tissue (e.g., via targetingtuning domains). For example, where the disease indicative protease isknown to be present in off-target sites but its presence in the targettissue is unexpected or indicative of disease, a delayed release of thatsubstrate might be desirable.

In various embodiments, polycyclic peptides may be used to increasesensitivity through the inclusion of 2, 3, 4, or more cyclic structureswhich may require the presence of different environmental conditions forlinearization or release of a functionalized reporter or therapeutic, orother molecule at a target location. For example, a bicyclic peptideaccording to certain embodiments is depicted in FIG. 8. The peptideincludes three engineered cleavage sites which must be cleaved in orderto release the functionalized molecule (in this case a glufib/K(CPQ₂)reporter) into the target environment. Two cyclization linkers areattached to cyclization residues on the peptide and such that threeenvironmentally sensitive cleavage substrates (in this case including aprotease-sensitive substrate cleavable at an aspartic acid residue) holdthe bicyclic peptide together and are required to be cleaved for releaseof the functionalized molecule. Any number of spacer residues may beused between the cyclization residues and the cleavage substrate inorder to achieve the desired peptide conformation and allow access tothe cleavage site by the target-specific protease or other cleavagemechanism. The substrates can be the same with the redundant cleavagerequirement further protecting from off-target or incidental release ofthe functionalized molecule. In certain embodiments, the varioussubstrates can be sensitive to different combinations of environmentalfactors or enzymes, serving as an and gate and thereby increasingspecificity.

Such sensitivity can be useful, for example, in instances whereoverexpression of a particular enzyme is indicative of disease only in acertain tissue but is normally present in other healthy tissues. Second,third, or more other cleavage substrates with different sensitivitiescan be included that are specific to the target tissue. Accordingly,only the presence of both the target-specific environmental cue and thetissue-specific, disease-specific environmental cue will release thereporter, reducing false positives.

FIG. 9 illustrates an exemplary cleavage of the bicyclic peptide in FIG.8. The bicyclic peptide is exposed to a protease specific to the threeengineered cleavage substrates and, after 3 cleavages, results in twoseparate molecules including separation of a fluorescent reporter from aquenching agent and mass tag. The polycyclic peptide concept can beexpanded to include 3, 4, or more cyclic structures with correspondinglygreater numbers of cleavages required for release of the reporter orother functionalized molecule. FIG. 10 shows a tricyclic peptiderequiring 4 cleavages for release of its reporter molecule. Of note, thereporter molecule or other functional molecule to be released inpolycyclic peptides having an odd number of cyclic structures (e.g., thetricyclic peptide shown in FIG. 10) is not located at a peptide terminusbut instead is positioned inside of the second cyclization residue froma terminus (e.g., the N-terminus in FIGS. 10 and 13). FIG. 11 shows apolycyclic peptide with 4 cyclic structures and 5 cleavage substratesrequiring cleavage to release its reporter molecule, which, having aneven number of cyclic structures and a corresponding odd number ofcleavage substrates, is located at the N-terminus. FIGS. 12 and 13 showpolycyclic peptides having n cyclic structures and n+1 correspondingcleavage structures. In FIG. 12 n is an even number while in FIG. 13 nis an odd number with the reporter molecule position varyingaccordingly.

FIG. 6 shows hydrolysis of the ester bond of a cyclic depsipeptidefollowed by cleavage of a protease-specific substrate to first linearizethe peptide and then release and activate a reporter molecule byseparating the quenching agent (CPQ₂) from the fluorescent reporter(FAM). The ester bond may be used to extend the half-life of thereporter molecule in blood.

When the cyclic peptide enters the diseased microenvironment, forexample tissues of a diseased liver or kidney, proteases with activityspecific to the linking substrates cleave the cyclic polypeptide,linearizing the peptide and liberating the reporter or therapeuticpeptide from the carrier.

Therapeutic peptides have been or are being developed to treat a widerange of conditions including applications in metabolic disease,oncology, and cardiovascular disease. See Lau and Dunn, 2018,Therapeutic peptides: Historical perspectives, current developmenttrends, and future directions, Bioorganic & Medicinal Chemistry26:2700-2707, incorporated herein by reference. Cyclization of suchtherapeutic peptides to block off-target reactions and protect againstdegradation during circulation and targeting can enhance theopportunities and applications of such therapeutic peptides whereengineered with a target-environment-specific cleavage site as describedherein.

Where the cyclic peptide harbors a reporter molecule, linearization andrelease of the reporter can occur when exposed to targetenvironment-specific protease or combination or proteases. The liberatedreporter may then re-enter circulation and pass through renal filtrationto urine or otherwise be excreted in any manner from the tissue and fromthe subject having the disease. The reporter may then be detected fromthe excreted sample in any suitable manner, for example, by massspectrometry or a ligand binding assay, such as an ELISA-based assay. Bydetecting the liberated reporter in the sample, the presence ofenzymatic activity upon the cyclic peptide is shown, thereby detectingthe target enzymatic activity.

The detected enzymatic activity may be activity of any type of enzyme,for example, proteases, kinases, esterases, peptidases, amidases,oxidoreductases, transferases, hydrolases, lysases, isomerases, orligases.

The biological sample may be any sample from a subject in which thereporter may be detected. For example, the sample may be a tissue sample(such as a blood sample, a hard tissue sample, a soft tissue sample,etc.), a urine sample, saliva sample, mucus sample, fecal sample,seminal fluid sample, or cerebrospinal fluid sample.

Reporter Detection

Where cleavage of the cyclic peptide releases a linearized reportermolecule, the reporter may be detected by any suitable detection methodable to detect the presence of quantity of molecules within thedetectable analyte, directly or indirectly. For example, reporters maybe detected via a ligand binding assay, which is a test that involvesbinding of the capture ligand to an affinity agent. Reporters may bedirectly detected, following capture, through optical density,radioactive emissions, nonradiative energy transfers. Alternatively,reporters may be indirectly detected with antibody conjugates, affinitycolumns, streptavidin-biotin conjugates, PCR analysis, DNA microarray,or fluorescence analysis.

A ligand binding assay often involves a detection step, such as anELISA, including fluorescent, colorimetric, bioluminescent andchemiluminescent ELISAs, a paper test strip or lateral flow assay, or abead-based fluorescent assay.

In one example, a paper-based ELISA test may be used to detect theliberated reporter in urine. The paper-based ELISA may be createdinexpensively, such as by reflowing wax deposited from a commercialsolid ink printer to create an array of test spots on a single piece ofpaper. When the solid ink is heated to a liquid or semi-liquid state,the printed wax permeates the paper, creating hydrophobic barriers. Thespace between the hydrophobic barriers may then be used as individualreaction wells. The ELISA assay may be performed by drying the detectionantibody on the individual reaction wells, constituting test spots onthe paper, followed by blocking and washing steps. Urine from the urinesample taken from the subject may then be added to the test spots, thenstreptavidin alkaline phosphate (ALP) conjugate may be added to the testspots, as the detection antibody. Bound ALP may then be exposed to acolor reacting agent, such as BCIP/NBT(5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt/nitro-bluetetrazolium chloride), which causes a purple colored precipitate,indicating presence of the reporter.

In another example, volatile organic compounds may be detected byanalysis platforms such as gas chromatography instrument, abreathalyzer, a mass spectrometer, or use of optical or acousticsensors.

Gas chromatography may be used to detect compounds that can be vaporizedwithout decomposition (e.g., volatile organic compounds). A gaschromatography instrument includes a mobile phase (or moving phase) thatis a carrier gas, for example, an inert gas such as helium or anunreactive gas such as nitrogen, and a stationary phase that is amicroscopic layer of liquid or polymer on an inert solid support, insidea piece of glass or metal tubing called a column. The column is coatedwith the stationary phase and the gaseous compounds analyzed interactwith the walls of the column, causing them to elute at different times(i.e., have varying retention times in the column). Compounds may bedistinguished by their retention times.

A modified breathalyzer instrument may also be used to detect volatileorganic compounds. In a traditional breathalyzer that is used to detectan alcohol level in blood, a subject exhales into the instrument, andany ethanol present in the subject's breath is oxidized to acetic acidat the anode. At the cathode, atmospheric oxygen is reduced. The overallreaction is the oxidation of ethanol to acetic acid and water, whichproduces an electric current that may be detected and quantified by amicrocontroller. A modified breathalyzer instrument exploiting otherreactions may be used to detect various volatile organic compounds.

FIG. 7 is a mass spectrum that may be used to detect a target activity,as described in step 125. Mass spectrometry may be used to detect anddistinguish reporters based on differences in mass. In massspectrometry, a sample is ionized, for example by bombarding it withelectrons. The sample may be solid, liquid, or gas. By ionizing thesample, some of the sample's molecules are broken into chargedfragments. These ions may then be separated according to theirmass-to-charge ratio. This is often performed by accelerating the ionsand subjecting them to an electric or magnetic field, where ions havingthe same mass-to-charge ratio will undergo the same amount ofdeflection. When deflected, the ions may be detected by a mechanismcapable of detecting charged particles, for example, an electronmultiplier. The detected results may be displayed as a spectrum of therelative abundance of detected ions as a function of the mass-to-chargeratio. The molecules in the sample can then be identified by correlatingknown masses, such as the mass of an entire molecule to the identifiedmasses or through a characteristic fragmentation pattern.

When the reporter includes a nucleic acid, the reporter may be detectedby various sequencing methods known in the art, for example, traditionalSanger sequencing methods or by next-generation sequencing (NGS). NGSgenerally refers to non-Sanger-based high throughput nucleic acidsequencing technologies, in which many (i.e., thousands, millions, orbillions) of nucleic acid strands can be sequenced in parallel. Examplesof such NGS sequencing includes platforms produced by Illumina (e.g.,HiSeq, MiSeq, NextSeq, MiniSeq, and iSeq 100), Pacific Biosciences(e.g., Sequel and RSII), and Ion Torrent by ThermoFisher (e.g., Ion S5,Ion Proton, Ion PGM, and Ion Chef systems). It is understood that anysuitable NGS sequencing platform may be used for NGS to detect nucleicacid of the detectable analyte as described herein.

Analysis may be performed directly on the biological sample or thedetectable analyte may be purified to some degree first. For example, apurification step may involve isolating the detectable analyte fromother components in the biological sample. Purification may includemethods such as affinity chromatography. The isolated or purifieddetectable analyte does not need to be 100% pure or even substantiallypure prior to analysis.

Detecting the detectable analyte may provide a qualitative assessment(e.g., whether the detectable analyte is present or absent) or aquantitative assessment (e.g., the amount of the detectable analytepresent) to indicate a comparative activity level of the enzymes. Thequantitative value may be calculated by any means, such as, bydetermining the percent relative amount of each fraction present in thesample. Methods for making these types of calculations are known in theart.

The detectable analyte may be labeled. For example, a label may be addeddirectly to a nucleic acid when the isolated detectable analyte issubjected to PCR. For example, a PCR reaction performed using labeledprimers or labeled nucleotides will produce a labeled product. Labelednucleotides, such as fluorescein-labeled CTP are commercially available.Methods for attaching labels to nucleic acids are well known to those ofordinary skill in the art and, in addition to the PCR method, include,for example, nick translation and end-labeling.

Labels suitable for use in the reporter include any type of labeldetectable by standard methods, including spectroscopic, photochemical,biochemical, electrical, optical, or chemical methods. The label may bea fluorescent label. A fluorescent label is a compound including atleast one fluorophore. Commercially available fluorescent labelsinclude, for example, fluorescein phosphoramidites, rhodamine,polymethadine dye derivative, phosphores, Texas red, green fluorescentprotein, CY3, and CY5.

Other known techniques, such as chemiluminescence or colormetrics(enzymatic color reaction), can also be used to detect the reporter.Quencher compositions in which a “donor” fluorophore is joined to an“acceptor” chromophore by a short bridge that is the binding site forthe enzyme may also be used. The signal of the donor fluorophore isquenched by the acceptor chromophore through a process believed toinvolve resonance energy transfer (RET), such as fluorescence resonanceenergy transfer (FRET). Cleavage of the peptide results in separation ofthe chromophore and fluorophore, removal of the quench, and generationof a subsequent signal measured from the donor fluorophore. Examples ofFRET pairs include 5-Carboxyfluorescein (5-FAM) and CPQ2, FAM andDABCYL, Cy5 and QSY21, Cy3 and QSY7.

In various embodiments, the cyclic peptide may include ligands to aid ittargeting particular tissues or organs. When administered to a subject,the cyclic peptide is trafficked in the body through various pathwaysdepending on how it enters the body. For example, if cyclic peptide isadministered intravenously, it will enter systemic circulation from thepoint of injection and may be passively trafficked through the body.

For the cyclic peptide to respond to enzymatic activity within aspecific cell, at some point during its residence time in the body, thecyclic peptide must come into the presence of the enzyme and have anopportunity to be cleaved and linearized by the enzyme to release thelinearized reporter or therapeutic molecule. From a targetingperspective, it is advantageous to provide the cyclic peptide with ameans to target specific cells or a specific tissue type where suchenzymes of interest may be present. To achieve this, ligands forreceptors of the specific cell or specific tissue type may be providedas the tuning domains and linked to polypeptide.

Cell surface receptors are membrane-anchored proteins that bind ligandson the outside surface of the cell. In one example, the ligand may bindligand-gated ion channels, which are ion channels that open in responseto the binding of a ligand. The ligand-gated ion channel spans thecell's membrane and has a hydrophilic channel in the middle. In responseto a ligand binding to the extracellular region of the channel, theprotein's structure changes in such a way that certain particles or ionsmay pass through. By providing the cyclic peptide with tuning domainsthat include ligands for proteins present on the cell surface, thecyclic peptide has a greater opportunity to reach and enter specificcells to detect enzymatic activity within those cells.

By providing the cyclic peptide with tuning domains, distribution of thecyclic peptide may be modified because ligands may target the cyclicpeptide to specific cells or specific tissues in a subject via bindingof the ligand to cell surface proteins on the targeted cells. Theligands of tuning domains may be selected from a group including a smallmolecule; a peptide; an antibody; a fragment of an antibody; a nucleicacid; and an aptamer.

Once cyclic peptide reaches the specific tissue, ligands may alsopromote accumulation of the cyclic peptide in the specific tissue type.Accumulating the cyclic peptide in the specific tissue increases theresidence time of the cyclic peptide and provides a greater opportunityfor the cyclic peptide to be enzymatically cleaved by proteases in thetissue, if such proteases are present.

When the cyclic peptide is administered to a subject, it may berecognized as a foreign substance by the immune system and subjected toimmune clearance, thereby never reaching the specific cells or specifictissue where the specific enzymatic activity can release the therapeuticcompound or reporter molecule. To inhibit immune detection, it ispreferable to use a biocompatible carrier so that it does not elicit animmune response, for example, a biocompatible carrier may include one ormore subunits of poly(ethylene glycol) maleimide. Further, the molecularweight of the poly(ethylene glycol) maleimide carrier may be modified tofacilitate trafficking within the body and to prevent clearance of thecyclic peptide by the reticuloendothelial system. Through suchmodifications, the distribution and residence time of the cyclic peptidein the body or in specific tissues may be improved.

In various embodiments, the cyclic peptide may be engineered to promotediffusion across a cell membrane. As discussed above, cellular uptake ofcyclic peptides has been well documented. See Gang. Hydrophobic chainsmay also be provided as tuning domains to facilitate diffusion of thecyclic peptide across a cell membrane may be linked to the cyclicpeptide.

The tuning domains may include any suitable hydrophobic chains thatfacilitate diffusion, for example, fatty acid chains including neutral,saturated, (poly/mono) unsaturated fats and oils (monoglycerides,diglycerides, triglycerides), phospholipids, sterols (steroid alcohols),zoosterols (cholesterol), waxes, and fat-soluble vitamins (vitamins A,D, E, and K).

In some embodiments, the tuning domains include cell-penetratingpeptides. Cell-penetrating peptides (CPPs) are short peptides thatfacilitate cellular intake/uptake of cyclic peptides of the disclosure.CPPs preferably have an amino acid composition that either contains ahigh relative abundance of positively charged amino acids such as lysineor arginine or has sequences that contain an alternating pattern ofpolar/charged amino acids and non-polar, hydrophobic amino acids. SeeMilletti, 2012, Cell-penetrating peptides: classes, origin, and currentlandscape, Drug Discov Today 17:850-860, incorporated by reference.Suitable CPPs include those known in the literature as Tat, R6, R8, R9,Penetratin, pVEc, RRL helix, Shuffle, and Penetramax. See Kristensen,2016, Cell-penetrating peptides as tools to enhance non-injectabledelivery of biopharmaceuticals, Tissue Barriers 4(2):e1178369,incorporated by reference.

In certain embodiments, a cyclic peptide may include a biocompatiblepolymer as a tuning domain to shield the cyclic peptide from immunedetection or inhibit cellular uptake of the cyclic peptide bymacrophages.

When a foreign substance is recognized as an antigen, an antibodyresponse may be triggered by the immune system. Generally, antibodieswill then attach to the foreign substance, forming antigen-antibodycomplexes, which are then ingested by macrophages and other phagocyticcells to clear those foreign substances from the body. As such, whencyclic peptide enters the body, it may be recognized as an antigen andsubjected to immune clearance, preventing the cyclic peptide fromreaching a specific tissue to detect enzymatic activity. To inhibitimmune detection of the cyclic peptide, for example, PEG tuning domainsmay be linked to the cyclic peptide. PEG acts as a shield, inhibitingrecognition of the cyclic peptide as a foreign substance by the immunesystem. By inhibiting immune detection, the tuning domains improve theresidence time of the cyclic peptide in the body or in a specifictissue.

Enzymes have a high specificity for specific substrates by bindingpockets with complementary shape, charge and hydrophilic/hydrophobiccharacteristic of the substrates. As such, enzymes can distinguishbetween very similar substrate molecules to be chemoselective (i.e.,preferring an outcome of a chemical reaction over an alternativereaction), regioselective (i.e., preferring one direction of chemicalbond making or breaking over all other possible directions), andstereospecific (i.e., only reacting on one or a subset ofstereoisomers).

Steric effects are nonbonding interactions that influence the shape(i.e., conformation) and reactivity of ions and molecules, which resultsin steric hindrance. Steric hindrance is the slowing of chemicalreactions due to steric bulk, affecting intermolecular reactions.Various groups of a molecule may be modified to control the sterichindrance among the groups, for example to control selectivity, such asfor inhibiting undesired side-reactions. By providing the cyclic peptidewith tuning domains such as spacer residues between the cyclizationlinker and residues and the cleavage site and/or any bioconjugationresidue, steric hindrance among components of cyclic peptide may beminimized to increase accessibility of the cleavage site to specificproteases. Alternatively, steric hindrance can be used as describedabove to prevent access to the cleavage site until an unstablecyclization linker (e.g., an ester bond of a cyclic depsipeptide) hasdegraded. Such unstable cyclization linkers can be other known chemicalmoieties that hydrolyze in defined conditions (e.g., pH or presence of acertain analyte) which may be selected to respond to specificcharacteristics of a target environment.

In various embodiments, cyclic peptides may include D-amino acids asidefrom the target cleavage site to further prevent non-specific proteaseactivity. Other non-natural amino acids may be incorporated into thepeptides, including synthetic non-native amino acids, substituted aminoacids, or one or more D-amino acids.

In some embodiments, tuning domains may include synthetic polymers suchas polymers of lactic acid and glycolic acid, polyanhydrides,polyurethanes, and natural polymers such as alginate and otherpolysaccharides including dextran and cellulose, collagen, albumin andother hydrophilic proteins, zein and other prolamines and hydrophobicproteins, copolymers and mixtures thereof.

One of skill in the art would know what peptide segments to include asprotease cleavage sites in a cyclic peptide of the disclosure. One canuse an online tool or publication to identify cleavage sites. Forexample, cleavage sites are predicted in the online database PROSPER,described in Song, 2012, PROSPER: An integrated feature-based tool forpredicting protease substrate cleavage sites, PLoSOne 7(11):e50300,incorporated by reference. Any of the compositions, structures, methodsor cyclic peptides discussed herein may include, for example, anysuitable cleavage site, as well as any further arbitrary polypeptidesegment to obtain any desired molecular weight. To prevent off-targetcleavage, one or any number of amino acids outside of the cleavage sitemay be in a mixture of the D and/or the L form in any quantity.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

What is claimed is:
 1. An engineered cyclic peptide comprising: one or more cleavage sites cleavable within a target environment and generally resistant to cleavage outside a target environment, wherein cleavage of the one or more cleavage sites releases a linearized peptide reactive with the target environment.
 2. The engineered cyclic peptide of claim 1 wherein the target environment is a tumor.
 3. The engineered cyclic peptide of claim 1 wherein the target environment is a biological fluid.
 4. The engineered cyclic peptide of claim 3 wherein the biological fluid is blood.
 5. The engineered cyclic peptide of claim 1 wherein the cleavage site is cleaved by an enzyme present in the target environment.
 6. The engineered cyclic peptide of claim 5 wherein the enzyme is known to be expressed with a certain disease or medical condition.
 7. The engineered cyclic peptide of claim 6 wherein the linearized peptide is a therapeutic peptide operable to treat the disease or medical condition.
 8. The engineered cyclic peptide of claim 1 wherein the linearized peptide is bioactive within the target environment.
 9. The engineered cyclic peptide of claim 1 wherein the linearized peptide is cleavable in response to pH of the target environment.
 10. The engineered cyclic peptide of claim 1 wherein the cyclic peptide is a cyclic depsipeptide and the one or more cleavage sites comprise an ester bond.
 11. The engineered cyclic peptide of claim 1 wherein the cyclic peptide is a macrocyclic peptide.
 12. The engineered cyclic peptide of claim 1 further comprising a carrier.
 13. The engineered cyclic peptide of claim 12 wherein the carrier comprises a poly ethylene glycol (PEG) scaffold of covalently linked PEG subunits.
 14. The engineered cyclic peptide of claim 1 wherein the linearized peptide is a detectable reporter
 15. The engineered cyclic peptide of claim 14 wherein the detectable reporter comprises one selected from the group consisting of: a volatile organic compound; an elemental mass tag; a peptide comprising one or more D-amino acids; a nucleic acid; and a neoantigen.
 16. The engineered cyclic peptide of claim 14 wherein the detectable reporter comprises an elemental mass tag comprising an element of atomic number greater than
 20. 17. The engineered cyclic peptide of claim 14 wherein the reporter comprises an antigen detectable by a hybridization assay.
 18. The engineered cyclic peptide of claim 1 wherein the one or more cleavage sites comprise a plurality of different cleavage sites.
 19. The engineered cyclic peptide of claim 18 wherein the plurality of different cleavage sites are cleaved by different enzymes.
 20. The engineered cyclic peptide of claim 18 wherein cleavage of two or more of the plurality of different cleavage sites is required to release the linearized peptide.
 21. The engineered cyclic peptide of claim 20 wherein the two or more of the plurality of different cleavage sites must be cleaved in a specific order to release the linearized peptide.
 22. The engineered cyclic peptide of claim 21 further comprising a tuning domain that modifies a distribution or residence time of the engineered cyclic peptide within a subject when administered to the subject.
 23. The engineered cyclic peptide of claim 22 further comprising a plurality of tuning domains wherein the tuning domains comprise ligands for receptors of a specific cell or a specific tissue type.
 24. The engineered cyclic peptide of claim 23 wherein the ligands promote accumulation of the engineered cyclic peptide in the specific tissue type or body compartment, wherein the ligands each comprise one selected from the group consisting of a small molecule; a peptide; an antibody; a fragment of an antibody; a nucleic acid; and an aptamer.
 25. The engineered cyclic peptide of claim 22 further comprising a plurality of tuning domains wherein the tuning domains comprise hydrophobic chains that facilitate diffusion of the engineered cyclic peptide across a cell membrane. 